Project Summary Mechanotransduction machinery located at the tips of hair bundles in the inner ear are responsible for our sensations of movement and sound. Each bundle is composed of ~100 stereocilia organized in a staircase array that are connected by tip-links, extracellular protein filaments composed of protocadherin 15 (PCDH15) and cadherin-23 (CDH23). Deflection of the hair bundle by sound and fluid movement leads to opening of the mechanotransduction channel (MEC) complex located at the lower tip-link insertion site, resulting in an electrical signal. There are three putative components of the MEC complex aside from the tip-link proteins: the lipoma HMGIC fusion partner-like 5 protein LHFPL5 (also known as TMHS), the transmembrane inner ear protein TMIE, and the transmembrane-like channel proteins TMC1/2, which are the likely pore-forming subunits of the complex. Although it is known that these proteins interact to form a mechanotransduction complex necessary for hearing, the precise composition, stoichiometry, and structure of this complex remain elusive. The goal of this proposal is to elucidate the composition and architecture of the MEC complex using single-molecule techniques and cryo- electron tomography. The main challenge of the proposed work is the low abundance of the MEC complex in native tissue. We have developed high affinity antibodies against three of the four MEC complex components that will be key elements in our approach to overcome this issue. In the first aim, I will utilize these antibodies to pull-down and detect the MEC complex from mouse cochlear hair cells using a single molecule pulldown method to assess MEC complex composition. Photobleaching and single-molecule quantitation experiments will allow me to determine MEC complex stoichiometry and measure the number of MEC complexes per cochlea. In the second aim of this proposal, I will elucidate the architecture of the MEC complex using cryo-electron tomography. I have developed a method to prepare cryo-EM grids with stereocilia in vitreous ice and obtained promising preliminary tomograms. I will use our antibodies conjugated to quantum dots to label the MEC complex on cryo- EM grids and determine a 3D structure of the complex. Illuminating the architecture and composition of the MEC complex will provide valuable insight into the elusive mechanism of hair cell mechanotransduction and open doors for the development of new therapeutics and treatment strategies for deaf individuals.